The invention provides isolated nucleotide and amino acid sequences of Slo3, a pH sensitive potassium channel expressed in sperm; antibodies to Slo3; methods if of screening for Slo3 inhibitors and activators; and methods of identifying Slo3 homologs.
Potassium channels are found in a wide variety of animal cells such as nervous, muscular, glandular, immune or epithelial tissue. The channels regulating these currents open and allow the escape of potassium under certain conditions. The outward flow of potassium ions upon opening of these channels makes the interior of the cell more negative, counteracting depolarizing voltages applied to the cell. These channels are regulated, e.g., by calcium sensitivity, voltage-gating, and ATP-sensitivity.
The Drosophila Slo1 gene encodes a calcium-activated potassium channel present in both neurons and muscle (Elkins et al., Proc. Natl. Acad. Sci. U.S.A. 83:8415 (1986); Atkinson et al., Science 253:551 (1991); and Adelman et al., Neuron 9:209 (1992)). Mammalian homologs of dSlo1 were cloned and found to be xe2x80x9cMaxixe2x80x9d or BK (large conductance) channel types, as the single channel conductance was 272 pS with symmetrical potassium concentrations. Slo1 channels cloned from mouse and human show strong conservation of sequence and functional properties (Butler et al., Science 261:221-224 (1993); Dworetzky et al., Brain Res. Mol. Brain Res. 27:189-193 (1994); Tseng-Crank et al., Neuron 13:1315-1330 (1994); McCobb et al., Am. J. Physiol. 269:H767-H777 (1995); and Wallner et al., Rec. Chan. 3:185-199 (1995)). One proposed role of the Slo1 channel is to provide negative feedback for the entry of calcium into cells via voltage-dependent calcium channels. Perhaps because of the versatility of this mechanism, Slo1 channels are expressed in many tissues, including brain, skeletal and smooth muscle, auditory hair cells, pancreas, and adrenal gland (Marty, Nature 291:497-500 (1981); Pallotta et al., Nature 293:471-474 (1981); Petersen and Mauryama, Nature 307:693-696 (1984); Tabcharani and Misler, Biochim. Biophys. Acta. 982:62-72 (1990); Neely and Lingle, J. Physiol. 453:97-131 (1992)). In these tissues, Slo1 channels are involved in diverse functions such as regulating arteriolar smooth muscle tone (Brayden and Nelson, Science 256:532-535 (1992)), tuning of hair cell frequency (Fuchs, Curr. Op. Neurobiol. 2:457-461 (1992); Wu et al., Prog. Biophys. Mol. Bio. 63:131-158 (1996)), and modulation of transmitter release at nerve terminals (Robitaille and Charlton, J. Neurosci. 12:297-305 (1995); Knaus et al., J. Neurosci. 16:955-963 (1996)), all situations in which both membrane potential and intracellular calcium are critical factors. While numerous family members of every type of voltage-gated K+ channel have been found, to date the Slo1 channel has remained the sole functionally characterized representative of the Slo family (Wei et al., Neuropharmacology 35:805-829 (1996)).
Spermatocytes require proteins tailored to fulfill roles unique to the process of germ cell development and fertilization. Cellular signaling in spermatic cells is tightly regulated to prevent inappropriate activation of the irreversible steps that prepare the sperm to fertilize the oocyte. Many of these steps are triggered and coordinated by changes in membrane potential and intracellular Ca2+ concentration and pH. Between mating and fertilization, sperm undergo capacitation, a process which later enables them to fertilize the oocyte. Capacitation involves an increase in cytosolic pH (pHi), which promotes metabolic and swimming activity (Babcock et al., Proc. Natl. Acad. Sci. USA 80:1327-1331 (1983); Babcock and Pfeiffer, J. Biol. Chem. 262:15041-15047 (1987); Vredenburgh-Wilberg and Parrish, Mol. Reprod. Dev. 40:490-502 (1995)). This increase in pHi is accompanied by changes in membrane potential and a rise in cytoplasmic Ca2+, which trigger the acrosome reaction upon contact with the oocyte (Amoult et al., J. Cell Biol. 134:637-645 (1996); Florman, Dev. Biol. 165:152-164 (1994)). Because of the central importance of these events in development, many efforts have been made to identify the specific proteins, including ion channels, which regulate spermatic function. In particular, there have been reports of channels present in spermatocytes and spermatids that have been proposed to play central roles in these reactions (Cook and Babcock, J. Biol. Chem. 268:22402-22407 (1993), including voltage dependent calcium channels (Florman et al., Dev. Biol. 152:304-214 (1992); Arnoult et al., Proc. Natl. Acad. Sci. USA 93:13004-13009 (1996); Lievano et al., FEBS Lett. 388:150-154(1996); Santi et al., Am. J. Physiol. 271:C1583-C1593 (1996)). Apart from a cyclic nucleotide gated channel, however, few of these channels have been directly cloned from testis (Weyand et al., Nature 368:859-863 (1994)).
Potassium channels have evolved to play specialized roles in many inexcitable tissues. The present invention provides for the first time isolated nucleotide and amino acid sequences of Slo3, a potassium channel with novel functional properties, abundantly expressed in spermatocytes. The physiological reactions that sperm undergo to achieve fertilization include changes in both pHi and membrane potential. Although Slo3 is a member of the Slo family, to which the large-conductance, calcium-activated Slo1 potassium channel belongs, Slo3 channels are not gated by calcium. Slo3 channels, however, are activated by changes in intracellular pH and membrane potential. Slo3 channels also exhibit markedly lower selectivity for K+ over Na+ than most voltage-gated K+ channels.
In one aspect, the invention provides an isolated nucleic acid encoding a polypeptide monomer of a pH sensitive potassium channel. The monomer (i) has a calculated molecular weight of between 120-156 kDa; (ii) has a unit conductance of approximately 80-120 pS when the monomer is in a functional tetrameric form of a potassium channel and is expressed in a Xenopus oocyte; (iii) has increased activity above approximately intracellular pH of 7.1; and (iv) specifically binds to polyclonal antibodies generated against SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:16 or SEQ ID NO:18.
In one embodiment, the nucleic acid encodes mSlo3 or hSlo3. In another embodiment, the nucleic acid encodes SEQ ID NO:1, SEQ ID NO:16, or SEQ ID NO:18. In one embodiment, the nucleic acid selectively hybridizes under moderate stringency hybridization conditions to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:17, or SEQ ID NO:19. In one embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:17, or SEQ ID NO:19.
In one embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as the primer sets selected from the group consisting of:
CTCGAACTCCCTAAAATCTTACAGAT (SEQ ID NO:8) and
TTCCGTTGAGCCAGGGGTCACCAGAATT (SEQ ID NO:9);
TCTGCTTTGTGAAGCTAAATCT (SEQ ID NO:10) and
TTTCAAAGCCTCTTTAGCGGTAA (SEQ ID NO:11); and
TTATGCCTGGATCTGCACTCTACATG (SEQ ID NO:12) and
ATAGTTTCCGTCTACTACCGAAA (SEQ ID NO:13).
In another embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as the primer sets selected from the group consisting of:
GGCAGCGCTCATTCTTTCCTCCTT (SEQ ID NO:14) and
TGCCCAAAACCTCAACCCAAAATA (SEQ ID NO:15).
In another aspect, the invention provides an isolated nuclcic acid encoding at least 15 contiguous amino acids from a pH sensitive potassium channel polypeptide monomer, said monomer having an amino acid sequence of SEQ ID NO:1, SEQ ID NO:16, or SEQ ID NO:18 and conservatively modified variants thereof.
In one embodiment, the nucleic acid encodes a pH sensitive potassium channel polypeptide monomer having: (i) a unit conductance of 80-120 pS when the monomer is in a functional tetrameric form of a potassium channel and is expressed in a Xenopus oocyte; (ii) a molecular weight of between 120-156 kDa; and (iii) increased activity above an intracellular pH of 7.1; and where the nucleic acid either: (i) selectively hybridizes under moderate stringency hybridization conditions to a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:17 or SEQ ID NO:19; or (ii) encodes a protein which could be encoded by a nucleic acid that selectively hybridizes under moderate stringency hybridization conditions to a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:17 or SEQ ID NO:19.
In another aspect, the invention provides an isolated nucleic acid encoding a polypeptide monomer of a pH sensitive potassium channel, the sequence: (i) encoding a monomer having a core domain that has greater than 60% amino acid sequence identity to amino acids 35-641 of an mSlo3 core domain as measured using a sequence comparison algorithm; and (ii) specifically binding to polyclonal antibodies raised against the core domain of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:16, or SEQ D NO:18.
In another aspect, the invention provides an isolated polypeptide monomer of a pH sensitive potassium channel, the monomer: (i) having a calculated molecular weight of between 120-156 kDa; (ii) having a unit conductance of approximately 80-120 pS when the monomer is in a functional tetrameric form of a potassium channel and is expressed in a Xenopus oocyte; (iii) having increased activity above approximately intracellular pH of 7.1; and (iv) specifically binding to polyclonal antibodies generated against SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:16, or SEQ ID NO:18.
In another aspect, the invention provides an antibody that selectively binds to mSlo3 or hSlo3.
In another aspect, the invention provides an expression vector comprising a nucleic acid encoding a polypeptide monomer of a pH sensitive potassium channel, the monomer: (i) having a calculated molecular weight of between 120-156 kDa; (ii) having a unit conductance of approximately 80-120 pS when the monomer is in a functional tetrameric form of a potassium channel and is expressed in a Xenopus oocyte; (iii) having increased activity above approximately intracellular pH of 7.1; and (iv) specifically binding to polyclonal antibodies generated against SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:16, or SEQ ID NO:18.
In another aspect, the invention provides a host cell comprising the expression vector.
In another aspect, the invention provides a method for identifying a compound that increases or decreases ion flux through a pH sensitive potassium channel, the method comprising the steps of: (i) contacting the compound with a eukaryotic host cell or cell membrane in which has been expressed a pH sensitive potassium channel monomer polypeptide: (a) having a calculated molecular weight of between 120-156 kDa; (b) having a unit conductance of approximately 80-120 pS when the monomer is in the functional tetrameric form of a potassium channel and is expressed in a Xenopus oocyte; and (c) specifically binding to polyclonal antibodies generated against SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:16, or SEQ ID NO:18; and (ii) determining the functional effect of the compound upon the cell or cell membrane expressing the pH sensitive potassium channel.
In one embodiment, the increased or decreased flux of ions is determined by measuring whole cell conductance. In one embodiment, the pH sensitive potassium channel monomer polypeptide is recombinant.
In another aspect, the invention provides method of detecting the presence of Slo3 in mammalian tissue, the method comprising the steps of: (i) isolating a biological sample from a patient; (ii) contacting the biological sample with a Slo3-specific reagent that selectively binds to Slo3; and, (iii) detecting the level of Slo3-specific reagent that selectively associates with the sample.
In one embodiment, the Slo3 specific reagent is selected from the group consisting of: Slo3 specific antibodies, Slo3 specific oligonucleotide primers, and Slo3 nucleic acid probes. In one embodiment, the sample is from a human.
In another aspect, the invention provides in a computer system, a method of screening for mutations of Slo3 genes, the method comprising the steps of: (i) receiving input of a first nucleic acid sequence encoding a pH sensitive potassium channel protein having a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:17, SEQ ID NO:19 and conservatively modified versions thereof; (ii) comparing the first nucleic acid sequence with a second nucleic acid sequence having substantial identity to the first nucleic acid sequence; and (iii) identifying nucleotide differences between the first and second nucleic acid sequences.
In one embodiment, the second nucleic acid sequence is associated with a disease state.
In another aspect, the invention provides in a computer system, a method for identifying a three-dimensional structure of Slo3 proteins, the method comprising the steps of: (i) receiving input of at least about 10 amino acids of an amino acid sequence of a pH sensitive potassium channel monomer or at least about 30 nucleotides of a nucleic acid encoding the protein, the protein having an amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:16, or SEQ ID NO:18, and conservatively modified versions thereof; and (ii) generating a three-dimensional structure of the protein encoded by the amino acid sequence.
In one embodiment, the amino acid sequence is a primary structure and said generating step includes the steps of: (i) forming a secondary structure from said primary structure using energy terms encoded by the primary structure; and (ii) forming a tertiary structure from said secondary structure using energy terms encoded by said secondary structure. In one embodiment, the generating step includes the step of forming a quaternary structure from said tertiary structure using anisotropy terms encoded by the tertiary structure. In one embodiment, the method further comprises the step of identifying regions of the three-dimensional structure of the protein that bind to ligands and using the regions to identify ligands that bind to the protein.